ABSTRACT While the development of addictive behaviors and the associated short- and long-term neuroadaptations after exposure to cocaine have been extensively studied, the mechanisms of cocaine addiction have not been fully elucidated. Using a novel method developed in our laboratory, we have isolated exosomes from the mouse brain and found in preliminary studies that chronic cocaine treatment increased levels of exosomes in the brain and changed the protein and lipid content of exosomes. Since cocaine has been shown to cause dysfunctions in the endosomal/autophagic/lysosomal system, we hypothesize that cocaine enhances exosome secretion in order to release accumulation of cellular materials caused by the disturbed degradation pathway. However, recent studies indicate that exosomes also serve as intercellular communication vehicles. Therefore, we suggest that exosome secretion is involved in the addiction process by transferring addiction-related proteins, lipids, and RNAs to target cells. We propose to test this hypothesis by characterizing the exosomes isolated from the brain of mice treated with cocaine or saline as control. Time course of changes in the level and cargo compositions of exosomes during non-contingent and contingent cocaine administration, followed by withdrawal (Aim 1), brain regional comparison of exosomal characteristics (Aim 2), and the direct effects of isolated exosomes on cocaine-related cellular and behavioral changes (Aim 3) would identify exosome- dependent and brain region specific mechanisms that underlie addictive behaviors. We have identified four exosomal populations, and in Aim 1 we propose to characterize the exosome populations affected by acute and chronic cocaine treatment. We will determine secretion levels of exosomes and analyze their cocaine- dependent proteomics, lipidomics, and RNA profiles. While global analyses of these molecules are our goal, a special focus will be given to BDNF, NMDA ?2, and gangliosides based on our preliminary results that have shown cocaine-induced changes in the levels of these molecules in exosomes. In Aim 2 we will identify brain regions and cellular changes involved in cocaine-induced secretion of exosomes containing cocaine-altered proteins, lipids, and RNAs in order to determine the role of exosomes in the induction, sensitization, or withdrawal phases of addiction. In Aim 3 we will evaluate roles of exosomes in cocaine addiction. The effects of exosomes derived from cocaine-treated mice, as well as, from in vitro cocaine-treated primary cultured cortical neurons and glial cells, on addiction-related molecular, cellular, and behavioral changes will be tested in nave mice. Future studies will examine whether some of the cocaine-induced changes in proteins, lipids, and RNAs, found in brain exosomes are also observed in cerebrospinal fluid and serum exosomes isolated from cocaine-addicted humans. The studies proposed here aim to give an insight into mechanisms behind the role of exosomes in cocaine addiction and may lead to finding new targets for addiction therapy and/or new biomarkers for cocaine addiction and withdrawal.